MicroRNAs (miRNAs) are an important component of gene regulation in higher eukaryotes, eliciting either decay or translational repression of target mRNAs. miRNAs are often dynamically expressed, and some contribute to regulation of spatio-temporal processes. Control of these master regulators thus represents a fundamental aspect of gene regulation. Although mechanisms of miRNA up- regulation have been explored, little is known about how miRNAs are down-regulated, a necessary facet of dynamic expression. It has been previously proposed that down-regulation of miRNAs is primarily mediated by dilution during cell growth and division. However, such a mechanism is not sufficient to explain rapidly decreased levels of several miRNAs that have been observed during numerous responses. To address this issue, this proposal has two specific aims. First, I intend to determine miRNA expression during the cell cycle using high-throughput sequencing of small RNAs isolated from synchronized cells (Aim 1). This analysis will allow me to determine the relative roles of miRNA decay and dilution during the cell cycle. Moreover, by identifying miRNAs involved in the cell cycle, this analysis will extend our understanding of their role in the cell cycle and oncogenesis. Second, using unstable miRNAs identified either in Aim 1 or previously during stimulation of macrophages, I will investigate the molecular mechanisms of miRNA decay (Aim 2). I will identify the responsible exonuclease(s) by performing knock-down experiments. Binding partners will then be identified by isolating native complexes and subsequent mass spectrometry. To identify miRNA instability motifs, mutations will be made across the entire miRNA, and the stabilities of these constructs will then be compared with that of the endogenous, wild-type miRNA. This analysis will be performed in conjunction with bioinformatic approaches to identify common motifs in unstable miRNAs. Alleviation of miRNA-mediated repression is as important as induction of this regulation, and thus understanding the mechanisms by which miRNAs are down-regulated is critical to dissecting gene regulation in multicellular organisms. Public Health Relevance: Cells have the same collection of genes, but what makes them different is which genes are turned on and which are turned off. Tiny RNAs, called microRNAs, help do this;when these RNA molecules are not present in the proper numbers-either too many or too few,-cancer can sometimes develop and/or the development of an organism can occur improperly. This proposal focuses on understanding how the correct levels of microRNAs are controlled and maintained.